The stenosis and even occlusion of coronary arteries caused by atherosclerosis is the recognized pathological basis of coronary atherosclerotic heart disease (CHD). The prevention and treatment of atherosclerosis are of positive significance for reducing the incidence of CHD. Longxue Tongluo Capsule (LTC) is a novel drug composed of total phenolic extracts from DR. In an atherosclerotic model of ApoE-deficient mice induced by a high-fat diet (HFD), administration of low, medium, and high doses of LTC resulted in significant reductions in the atherosclerotic lesion area of the aortic sinus by 26.4% (p < 0.05), 30.1% (p < 0.05), and 46.5% (p < 0.01), respectively (Zheng et al., 2016a). However, Compared to ezetimibe, LTC did not demonstrate a significant effect on the improvement of blood lipid levels in this study. In hypercholesterolemic rats induced by a HFD, LTC exhibited a significant impact on regulating blood lipid levels. It notably reduce serum total cholesterol, triglycerides, and low-density lipoprotein cholesterol, while simultaneously increase high-density lipoprotein cholesterol levels (p < 0.001) (Zhou et al., 2018). In addition, the pathological examination of aortic tissue showed that LTC reduced the accumulation of atherosclerotic lesions due to mild diffuse thickening and mild multifocal fragmentation of elastic and collagen fibers in media. The varying effects of LTC on blood lipid levels observed in the two models may be attributed to factors, including the inherent differences between the models, the complexity of the drug’s pharmacological effects, and the species-specific responses to LTC. Future research should investigate these discrepancies to enhance our understanding of LTC’s mechanisms. Additionally, regarding the specific mechanisms by which LTC reduces atherosclerotic lesions, particularly its influence on elastic and collagen fibers, further studies are necessary to elucidate the underlying mechanisms and signaling pathways.

Vascular smooth muscle cell migration and proliferation are key processes in the neointima formation that occurs during atherosclerosis, and these events can be induced by platelet-derived growth factor (PDGF) (Won et al., 2011). Research has demonstrated that 2 μg/mL of LTC can significantly inhibit the migration of A7r5 cells induced by PDGF-BB (20 ng/mL) (p < 0.05) and markedly reduce the accumulation of Ca²⁺ in these cells (Zhou et al., 2018). When PDGF-BB binds to cell surface receptors, the phospholipase C-γ signaling pathway is activated. Phospholipase C-γ can catalyze the hydrolysis of phosphatidylinositol - 4, 5 - bisphosphate into inositol - 1, 4, 5 - trisphosphate and diacylglycerol. Inositol - 1, 4, 5 - trisphosphate subsequently interacts with its receptor on the endoplasmic reticulum, leading to the release of calcium from intracellular stores and a consequent increase in cytosolic Ca²⁺ concentration (Woll and Van Petegem, 2022; Huang et al., 2024). The elevated intracellular Ca²⁺ levels can activate calcium/calmodulin-dependent protein kinase (CaMK) (Najar et al., 2021). CaMK phosphorylates various transcription factors, including CREB (cAMP response element-binding protein), thereby promoting the expression of cell cycle-related genes such as cyclin D1, which facilitates cell cycle progression from G0/G1 to S phase (Liu et al., 2013). When A7r5 cells were treated with a calcium chelating to reduce intracellular Ca²⁺ levels, it was observed that PDGF-BB-induced cell proliferation was significantly inhibited. This finding indicates that increased intracellular Ca²⁺ levels are essential for the PDGF-BB-induced proliferation of A7r5 cells (Figure 1). In addition, during cell migration, cytoskeletal rearrangement plays a crucial role. Elevated intracellular Ca²⁺ levels triggered by PDGF-BB stimulation can activate small GTPases, particularly members of the Rho family (Vazão et al., 2011). These proteins can regulate the polymerization and depolymerization, altering cell morphology and motility.

Based on these findings, it is hypothesized that LTC may disrupt PDGF-BB signal transduction by lowering intracellular Ca²⁺ levels. This disruption in signal transduction ultimately inhibits the proliferation of A7r5 cells induced by PDGF-BB (20 ng/mL). MAPKs is an important pathway involved in cell proliferation, which may be related to the proliferation of A7r5 cells induced by PDGF-BB (Figure 1). However, the current understanding of these mechanisms is incomplete. More detailed and specific studies are necessary to elucidate how LTC regulates the signaling pathways associated with intracellular Ca²⁺ levels and to determine whether other targets or pathways are involved in the regulation of cell proliferation and migration. This understanding is essential for a comprehensive grasp of the role of LTC in the prevention and treatment of atherosclerosis.

CHD represents a significant global public health challenge. The exploration of new therapies is crucial for its prevention and treatment. DR, the primary component of Compound Longxuejie Capsule (CLC), has been the focus of many studies aimed at evaluating its efficacy in treating coronary heart disease.

In the treatment of patients with acute myocardial infarction, the CLC, when used as an adjunct to conventional Western medicine, can reduce the left ventricular end-diastolic diameter, improve the levels of myocardial injury markers (Bao et al., 2015), enhance the overall clinical treatment effect (Li et al., 2014), and improve blood viscosity in the patient’s hemorheology (Wang, 2003). In the animal experiment, total flavonoids from DR can counteract the changes observed in the J point and T wave of the electrocardiogram in rats with myocardial ischemia. Additionally, it can reduce the infarcted myocardial area and the incidence of ST segment elevations in dogs subjected to coronary artery ligation, while also lowering relevant serum indicators such as creatine kinase (CK), lactate dehydrogenase (LDH), and lactate (Fang et al., 2005). In rabbits with acute myocardial infarction, DR demonstrates a protective effect on left heart function, mitigates the disorder of myocardial tissue structure, slows the heart rate, and stabilizes heart function indicators. It includes preventing significant decreases in left ventricular systolic pressure, the maximum rate of rise of left ventricular pressure (+dp/dtmax), and the maximum rate of fall of left ventricular pressure (-dp/dtmax), as well as avoiding a significant increase in left ventricular end-diastolic pressure (Cheng et al., 2017a). When DR was used to intervene in the mouse model of myocardial infarction, metabolomic analysis revealed significant alterations in the levels of seven myocardial infarction metabolic markers: phytosphingosine, sphinganine, acetylcarnitine, cGMP, cAMP, L-tyrosine, and L-valine (Qi et al., 2013). These findings suggest that DR may improve outcomes in myocardial infarction by regulating vascular smooth muscle contraction, sphingolipid metabolism, phenylalanine metabolism, and branched-chain amino acid metabolism (Figure 2). Su Xin investigated the mechanism of the CLC in a rat model of myocardial infarction using network pharmacology and quantitative real - time polymerase chain reaction (Su X. et al., 2024). The results indicated that the PI3K/Akt pathway plays a crucial role in the anti-myocardial ischemia effects of the CLC. In patients with stable coronary heart disease, the CLC demonstrated superior efficacy in alleviating angina pectoris and improving electrocardiogram outcomes compared to the control group (Qing et al., 2009), while also enhancing the patients’ hemorheological status (Jiang et al., 2015). A randomized, double-blind, parallel controlled, multi-center clinical trial involving 418 patients showed that CLC is effective and safe in the treatment of stable angina pectoris (Qing et al., 2009).

It is important to note that the CLC is a compound formulation comprising multiple ingredients. Apart from DR, it includes various other traditional Chinese medicine components or excipients, such as panax notoginseng and borneol. The presence of these additional ingredients may influence the research outcomes. From the perspective of traditional Chinese medicine, DR and panax notoginseng are both blood circulation-activating drugs. Research showed that panax notoginseng protected human umbilical vein endothelial cells from H₂O₂-induced oxidative stress. The mechanism is inhibiting early cell apoptosis via upregulating vascular endothelial growth factor A (VEGFA) mRNA expression (Dong et al., 2019). In addition, panax notoginseng could improve angiogenesis in rats with myocardial infarction and hypoxic human coronary artery endothelial cells, also affect the level of miR200a promoter methylation and miR200a and VEGF molecular pathways (Wang J. et al., 2024). For borneol, its cardiovascular benefits include reducing atherosclerosis by inhibiting foam cell formation through its metabolites (He et al., 2024). These therapeutic effects are of critical importance in the treatment of CHD. Consequently, it should be recognized that the effects observed from the CLC in studies are not entirely representative of the effects attributed to DR. To mitigate the potential interference of other components on research findings, studies investigating the effects of DR should ideally utilize it in isolation to enhance the accuracy and reliability of the results. Furthermore, while current research indicates certain therapeutic benefits, there remains a need for more extensive, multi-center clinical trials to further validate its safety and efficacy.

The mechanisms underlying myocardial ischemia-reperfusion injury (MIRI) are complex and involve a variety of physiological and pathological processes. These processes include oxidative stress, disruptions in energy metabolism, myocardial cell apoptosis, calcium overload, autophagy, cell infiltration, and vascular endothelial dysfunction. These pathological mechanisms can lead to significant damage to myocardial cells.

Research has demonstrated that total flavonoids from DR can reduce the concentration of LDH in the culture medium of a hypoxic/reoxygenated neonatal rat cardiomyocyte model, thereby enhancing cell viability (Deng et al., 2006). In animal experiments, these flavonoids exhibit a protective effect in rats subjected to MIRI, as they can decrease the area of myocardial infarction and the apoptosis rate of cardiomyocytes, while also reducing serum levels of CK, creatine kinase MB (CK-MB), and LDH to some extent (Liang et al., 2017). Similar findings were observed in experiments involving New Zealand rabbits with MIRI. Total flavonoids from DR were shown to improve myocardial tissue condition, stabilize the structure of myocardial cell membranes, reduce the leakage of myocardial CK-MB, and enhance myocardial systolic function. CK, CK-MB, and LDH serve as markers for cardiomyocyte necrosis (Cheng et al., 2017b). During MIRI, the release of these markers in blood correlates positively with the extent of myocardial necrosis and reflects the severity of MIRI damage. Collectively, these findings indicated that total flavonoids from DR could lower serum levels of CK, CK-MB, and LDH in animal models of MIRI, demonstrating a significant protective effect on cardiomyocytes in MIRI animals. Further research suggested that this protective effect may be closely associated with the JAK2/STAT3 signaling pathway (Cheng, 2017).

High mobility group box 1 protein (HMGB1) serves as an ancillary transcription factor within the cell nucleus, interacting with DNA and various transcription factors to regulate gene transcription. When cells are damaged or stimulated, HMGB1 can be released extracellularly, functioning as a significant inflammatory mediator. Extracellular HMGB1 can activate diverse immune cells, including macrophages and neutrophils, prompting them to release inflammatory factors and subsequently initiate an inflammatory response. In the myocardial cells of the ischemia/reperfusion (I/R) rat model, irregular cell arrangement is observed, with the intercellular matrix exhibiting signs of edema and some cells displaying necrosis. Additionally, infiltration of eosinophils and neutrophils is evident. Pretreatment of the I/R rat model with total flavonoids from DR could ameliorate these conditions and significantly reduce the expression of HMGB1 and PI3K proteins in myocardial tissue (Su L. et al., 2024). So, the mechanism through which the total flavonoids from DR alleviate MIRI may be associated with the antagonism of the HMGB1/PI3K signaling pathway and the subsequent reduction of the inflammatory response.

Apoptosis of cells plays a crucial role in the mechanism of ischemia-reperfusion injury. The PI3K/AKT signaling pathway regulates various cellular activities, including apoptosis. Caspase-3 protein is a key executor of cell apoptosis. Experimental evidence demonstrated that the activation of the PI3K/AKT signaling pathway could reduce the expression of caspase-3 protein (Zhou et al., 2023). Meanwhile, the expression of anti-apoptotic proteins such as Bcl-2 and Bcl-xL was promoted and the activity of the pro-apoptotic protein Bax was inhibited. This suggested that the activation of the PI3K/AKT pathway may inhibit the apoptosis of myocardial cells during MIRI, thereby exerting a protective effect. In tree shrews with DR reperfusion after myocardial ischemia, it also has been observed that the expression of Bcl-2 protein in myocardial tissue while the downregulating of Bax protein levels. Concurrently, the expressions of GRP78, CHOP-1, p-P38, p-JNK-1, and p-ERK proteins were also inhibited (Yang et al., 2021). GRP78 serves as a representative protein of endoplasmic reticulum stress, while CHOP-1 is a key transcription factor involved in endoplasmic reticulum stress-induced apoptosis. The proteins p-P38, p-JNK-1, and p-ERK belong to the MAPK family and play significant roles in the regulation of apoptosis. Therefore, reperfusion of the myocardium with DR can reduce cell apoptosis, inhibit endoplasmic reticulum stress to a certain extent, and ultimately achieve a protective effect against MIRI. Further research showed that DR inhibited endoplasmic reticulum-induced apoptosis of myocardial cells may by regulating the miR-423-3p/ERK signaling pathway in the tree shrew myocardial I/R model (Yang et al., 2019).

Pyroptosis is a newly defined programmed cell death mode characterized by the formation of pores in the cell membrane, leading to an imbalance in intracellular and extracellular osmotic pressure and ultimately resulting in cell lysis. The process releases pro-inflammatory cytokines such as IL-1β and IL-18, which contribute to inflammatory response. The Caspase-3/GSDME pathway is one of the molecular mechanisms underlying pyroptosis. When activated, this signaling pathway induces pyroptosis in cardiomyocytes (Zheng et al., 2020). The study has shown that total flavonoids from DR can reduce serum levels of CK-MB and LDH in I/R rats, while also exerting a protective effect on myocardial cells subjected to I/R injury (Huang et al., 2022). Specifically, these flavonoids mitigate the loss of myocardial cell nucleus, decrease the area of myocardial infarction, reduce the degree of myocardial tissue disarray, and limit the infiltration of inflammatory cells in the myocardial interstitial. Notably, these protective effects coincide with a reduction in the expression levels of caspase-3, GSDME, and IL-1β mRNA. It suggests that inhibition of the Caspase-3/GSDME signaling pathway can partially counteract the pyroptosis induced by MIRI.

Some researchers have observed in the MIRI rat model that the total flavonoids from DR can upregulate the expression of Wnt2 protein, increase serum VEGF levels, and downregulate β-catenin protein expression (Liang et al., 2023). Wnt2 primarily functions to promote cell proliferation, while β-catenin serves as a transcription factor that regulates downstream genes associated with apoptosis. This suggests that the total flavonoids from DR may enhance Wnt2 expression and negatively regulate β-catenin expression, thereby promoting angiogenesis and mitigating myocardial cell damage resulting from MIRI.

In summary, DR can improve cardiac function-related indicators, such as CK, CK-MB, and LDH, in various animal species MIRI models. The primary protective effects of DR include the inhibition of inflammation, apoptosis, cell pyroptosis, and the promotion of angiogenesis. The mechanisms involved are linked to the JAK2/STAT3, HMGB1/PI3K, PI3K/AKT, Caspase-3/GSDME, and Wnt2/β-catenin signaling pathways. DR consistently demonstrates protective effects against MIRI in different animal models and methodologies, highlighting its therapeutic value (Figure 3).

Increased fibrinogen (Jay et al., 1991), erythrocyte aggregation (Schechner et al., 2005), whole blood viscosity (Kanakaraj and Singh, 1989) plasma viscosity (Leonhardt et al., 1977), decreased osmotic fragility and deformability (Kanakaraj and Singh, 1989) are hemorheological factors associated with cardiovascular disease. These factors can significantly increase the risk of thrombosis, promote the occurrence of cardiovascular disease, and aggravate the progression. It's been suggested in the literature that DR exhibited clinical antithrombosis efficacy similar to low molecular weight heparin (Liang et al., 2021). Zheng Jiao found that treating hyperlipidemia mice with LTC caused beneficial changes in hemorheology, including a reduction in whole blood viscosity. Still, the deformation curve integral area of the erythrocyte also increased and the improvement of osmotic fragility (Zheng et al., 2016b).

When blood vessels are damaged, tissue plasminogen activator (t-PA) can rapidly bind to specific regions of fibrin via its lysine residues. This binding subsequently activates plasminogen that is closely associated with fibrin, converting it into active plasmin. Plasmin, a potent hydrolase, can cleave polymerized fibrin within thrombi and transform it into soluble, low-molecular-weight products. The process could lead to the disruption and dissolution of the thrombus structure. Similarly, urokinase-type plasminogen activator (u-PA) exerts antithrombotic effects by promoting plasminogen activation, facilitating thrombolysis, and enhancing microcirculation through analogous mechanisms. Plasminogen activator inhibitor-1 (PAI-1) is a secreted single-chain glycoprotein and a member of the serine protease inhibitor family. In the fibrinolytic system, PAI-1 serves as the natural and specific inhibitor of both uPA and tPA. Abnormal expression of PAI-1 can disrupt the body’s coagulation and anticoagulation systems, potentially leading to thrombotic diseases and an increased risk of cardiovascular events. Therefore, inhibiting the abnormal expression of PAI-1 contributes to reducing the formation of thrombotic events. Loureirin B is an important flavonoid compound extracted from DR. Based on the plasma fibrin experiment, researchers employed the substrate chromogenic method to assess the inhibitory effect and dose dependence of Loureirin B on PAI-1, in addition to conducting a plasma fibrin experiment (Yan, 2016). The results indicated that Loureirin B effectively inhibits PAI-1, prevents the formation of the complex between PAI-1 and uPA, and exhibits a dose-dependent effect (Figure 4). Findings from the plasma fibrin experiment revealed that the activity of PAI-1 is inversely proportional to the concentration of Loureirin B inhibitor, whereas the activity of uPA is directly proportional. Consequently, the rate of fibrin dissolution in blood increases, demonstrating a significant antithrombotic effect. Figure 5A.

Nuclear factor kappa B (NF-κB) is a significant transcription factor that plays a crucial role in various physiological and pathological processes. In the inflammatory response, NF-κB serves as one of the primary regulatory factors. It can induce the expression of genes encoding various inflammatory mediators, such as TNF-α, IL-1β, IL-6, and IL-8, thereby facilitating the recruitment and activation of inflammatory cells and the releasing of inflammatory mediators (Figure 6). Research indicates that LTC treatment can decrease the number of NF-κB-positive stained microvessels in the aortic tissue of atherosclerotic rats. It suggests that LTC exerts an inhibitory effect on the activation and expression of NF-κB (Zhou et al., 2018). Additionally, Huang Biaohua and colleagues have demonstrated that the total flavonoids from DR can reduce the serum levels of inflammatory factors TNF-α and IL-6 in rats suffering from MIRI. Quantitative real - time polymerase chain reaction analysis reveals that the mRNA expression levels of TNF-α and IL-6 are downregulated (Huang et al., 2021).

It was reported that lipopolysaccharide (LPS)-induced human aortic smooth muscle cells (HASMC) proliferation and inflammation lead to the development of atherosclerosis (Yang et al., 2005). HASMC and macrophages activated by LPS secrete various inflammatory mediators, including TNF-α, IL-1β, IL-6, and IL-8 (Liu et al., 2007; Heo et al., 2008). Sook-Kyoung Heo found that the ethylacetate extract from DR can inhibit the production of TNF-α, IL-8, and IL-6 in LPS-treated HASMC and RAW264.7 macrophages (Heo et al., 2010). This inhibition is accompanied by a decrease in reactive oxygen species (ROS) levels. Notably, when RAW264.7 cells were pretreated with the NADPH oxidase assembly inhibitor AEBSF to inhibit ROS production before LPS stimulation, it was observed that the activation of MAPKs was suppressed, resulting in a simultaneous reduction of inflammatory factor levels. This suggested that the mechanism by which ethyl acetate extract from DR decreases inflammatory factor levels is associated with ROS-mediated activation of MAPKs.

Zhu Qianwei concluded through network pharmacology and molecular docking methods that the active ingredients in the CLC bind to targets such as AKT1 and PTGS2, influencing inflammatory signaling pathways (NF-κB and PPAR), thereby contributing to the treatment of CHD (Zhu et al., 2023). Researchers have identified that disorders in the IL-6-JAK/STAT3 pathway and the PI3K-AKT-mTOR pathway may represent significant pathological mechanisms underlying acute myocardial infarction. DR demonstrates a synergistic cardioprotective effect by regulating these two pathways and the expression of targets (VEGF, COX2, and PPARγ) (Li et al., 2018). Figure 5B.

Lipid peroxidation, which refers to the oxidative degradation of lipids resulting in cell membrane damage, is considered a predictive biomarker for atherosclerosis and cardiovascular diseases (Rao et al., 2011). Superoxide dismutase (SOD) and malondialdehyde (MDA) are both biomarkers closely associated with oxidative stress. MDA is primarily one of the final products of lipid peroxidation reactions in living organisms and a direct indicator of myocardial lipid peroxidation damage (Figure 7). It has been reported that lipid accumulation and I/R can cause cellular lipid peroxidation damage (Wang et al., 2009; Han et al., 2018). LTC has been shown to reduce MDA levels in the serum of hyperlipidemic ApoE^−/− mouse models subjected to a high-fat diet (Zheng et al., 2016b). In their research, Yang Tianrui and colleagues discovered that, in addition to decreasing MDA levels in the myocardial tissue of tree shrews with I/R injury, DR also significantly promotes an increase in SOD (Yang et al., 2018). However, the role of these biomarkers in cellular redox balance and their impact on the function and survival of cardiomyocytes has not been thoroughly investigated. Therefore, future studies must elucidate the mechanisms by which DR alleviates oxidative stress to achieve cardiovascular protection, which is significant for the development of its potential pharmacological effects.

LDH is a glycolytic enzyme that is widely present in human cells (Figure 7). Under conditions of oxidative stress, the intracellular redox balance becomes disrupted, leading to the attack of excessive free radicals on the active center, amino acid residues, and coenzyme binding sites of LDH. This disruption results in altered LDH activity. The total flavonoids from DR have been shown to reduce LDH concentrations in the culture medium of a neonatal rat cardiomyocyte hypoxia/reoxygenation model, while also enhancing the viability of cardiomyocytes damaged by Na₂S₂O₄ stimulation (Deng et al., 2006). Typically, mild oxidative stress may induce a compensatory increase in LDH activity to satisfy the energy metabolism demands of cells under stress. However, as oxidative stress intensifies, LDH activity may be inhibited due to excessive oxidative damage that compromises the protein structure and function of LDH. The observation that LDH concentration decreased rather than increased in the aforementioned study suggests that the oxidative stress experienced by cardiomyocytes stimulated by Na2S2O4 is severe. Figure 5C.

The increased adhesiveness of endothelial cells, resulting from impaired endothelial function, is the initial step in the formation of atherosclerosis. Zheng Jiao stimulated human umbilical vein endothelial cells with oxidized low-density lipoprotein and observed that the adhesion of these cells to monocytes significantly increased. Notably, LTC can significantly reverse this phenomenon, which is closely related to the regulation of the MAPK/IKK/IκB/NF-κB signaling pathway (Zheng et al., 2016a). Endothelial cells are primary producers of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and they secrete chemotactic factors like monocyte chemotactic protein-1 (MCP-1) (Figure 8). These molecules play pivotal roles in the progression of atherosclerosis. Mediated by ICAM-1 and VCAM-1, the adherence of white blood cells to vascular endothelial cells directly impairs endothelial integrity or induces endothelial dysfunction. This process facilitates the migration of white blood cells toward the vascular endothelium and stimulates the proliferation of vascular smooth muscle cells. Concurrently, the emergence of MCP-1 attracts monocytes within the bloodstream, causing them to adhere to the vascular endothelium and migrate beneath it. These monocytes differentiate into macrophages, engulfing cholesterol to form foam cells. Ultimately, this sequence of events leads to the development of atherosclerosis. The study found that a 4-week LTC intervention can reduce the serum levels of VCAM-1, ICAM-1, and MCP-1 in atherosclerotic model rats (Zhou et al., 2018). This result suggests that LTC may influence the expression of ICAM-1, VCAM-1, and MCP-1 through specific potential action pathways (such as the NF-κB signaling pathway), thereby playing a beneficial role in the prevention and treatment of atherosclerosis. These findings provide a vital research direction for further exploration of the specific mechanisms by which LTC may prevent and treat atherosclerosis. Figure 5D.

In the MIRI model, Western blot analysis demonstrated a significant increase in the expression of fibrosis-related proteins, including fibronectin, collagen I, collagen III, and α-SMA, in the cardiac tissue of MIRI mice (Figure 9). Following a 4-week intervention with Loureirin B, the elevated levels of these proteins were effectively reduced, indicating that Loureirin B treatment can significantly mitigate collagen fiber deposition induced by MIRI (Kong and Zhao, 2022). Particularly, Loureirin B also inhibited the expression of pro-fibrotic factors such as PAI-1, transforming growth factor-β1 (TGF-β1), TGF-β1 receptor, and phosphorylated Smad2/3. TGF-β1 is known to play a crucial role in the occurrence and progression of myocardial fibrosis, while Smad proteins serve as key downstream effectors in the TGF-β1 signaling pathway. Increasing evidence suggests that PAI-1 is involved in the pathogenesis of various fibrosis-related diseases, including liver, lung, and kidney fibrosis (Oda et al., 2001; Wang et al., 2007; Courey et al., 2011). Thus, inhibiting the PAI-1/TGF-β1/Smad signaling pathway may provide a promising strategy for alleviating myocardial fibrosis induced by MIRI (Kong and Zhao, 2022). Additionally, Pin1 has been implicated in fibrotic diseases, including cardiac fibrosis, and can modulate the production of TGF-β1 (Wu et al., 2017). The study found that Loureirin B can also ameliorate cardiac fibrosis both in vitro and in vivo via the Pin1/TGF-β1 signaling pathway (Cheng et al., 2022). Figure 5E.